Methods for the prevention or treatment of no-reflow following ischemia/reperfusion injury

ABSTRACT

The invention provides methods of preventing or treating cardiac ischemia-reperfusion injury in a mammalian subject. The methods provide administering aromatic-cationic peptides in effective amounts to prevent or treat an anatomic zone of no re-flow in mammalian subjects. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to subjects in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication Ser. Nos. 61/363,129, filed Jul. 9, 2010; 61/363,133, filedJul. 9, 2010; and 61/412,655, filed Nov. 11, 2010, the entiredisclosures of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present technology relates generally to compositions and methods ofpreventing or treating ischemia/reperfusion tissue injury. Inparticular, embodiments of the present technology relate toadministering aromatic-cationic peptides in effective amounts to preventor treat an anatomic zone of no re-flow in mammalian subjects, at riskfor, or suffering from, ischemia/reperfusion tissue injury.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art.

After acute myocardial infarction (AMI), the immediate therapeutic goalis to establish patency of the infarct-related artery. The successfulrestoration of epicardial coronary artery patency, however, does notnecessarily translate into improved tissue perfusion. Structuraldisruption or obstruction of the microvasculature, the so-called“no-reflow” phenomenon, could occur before or because of percutaneouscoronary intervention (PCI) and can impair coronary flow. Patients withthe no re-flow phenomenon have a poor clinical prognosis. The no re-flowphenomenon is generally associated with the presence of an anatomic zoneof no re-flow in the tissue. Advances in imaging modalities haveimproved visualization of no re-flow, showing its frequency to be higherthan was estimated by clinical judgment alone. This phenomenon isimportant because it correlates with infarct size and provides usefulprognostic information. No re-flow is associated with reduced leftventricular ejection fraction, left ventricular remodeling, and poorclinical outcomes, placing patients with this effect in a high-riskgroup among reperfused patients. Attention has shifted, therefore, awayfrom merely achieving epicardial artery patency and towards the statusof the microvasculature which can lead to anatomic zones of no re-flow.

SUMMARY

The present technology relates to the treatment or prevention of cardiacischemia-reperfusion injury in mammals through administration oftherapeutically effective amounts of aromatic-cationic peptides, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt, to subjects inneed thereof. In some embodiments, the present technology relates tomethod useful in the treatment or prevention of an anatomic zone ofno-reflow, zone of hemorrhage and infarct size followingischemia/reperfusion.

In some aspects, the disclosure provides a method of treating orpreventing an anatomic zone of no re-flow, comprising administering to asubject in need thereof a therapeutically effective amount of anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,e.g, D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt. In someembodiments, the method further comprises performing a revascularizationprocedure on the subject. In some embodiments, the aromatic-cationicpeptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to P_(t)+1, except that when a is 1,P_(t) may also be 1. In particular embodiments, the subject is a human.

In some embodiments, 2p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges. In some embodiments, the peptide comprises oneor more non-naturally occurring amino acids, for example, one or moreD-amino acids. In some embodiments, the C-terminal carboxyl group of theamino acid at the C-terminus is amidated. In certain embodiments, thepeptide has a minimum of four amino acids. The peptide may have amaximum of about 6, a maximum of about 9, or a maximum of about 12 aminoacids.

In some embodiments, the peptide comprises a tyrosine or a2′,6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ or2′,6′-Dmt-D-Arg-Phe-Lys-NH₂. In another embodiment, the peptidecomprises a phenylalanine or a 2′,6′-dimethylphenylalanine residue atthe N-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In a particularembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof such as acetate salt or trifluoroacetate salt.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered orally, topically,intranasally, intraperitoneally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis). In some embodiments, thearomatic-cationic peptide is administered by an intracoronary route oran intra-arterial route.

In one embodiment, the present technology provides a method forpreventing or treating anatomic zone of no re-flow in a mammaliansubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as acetate salt or trifluoroacetate salt, therebypreventing or treating microvasular injury in the subject. In oneembodiment, the method further comprises the step of performing arevascularization procedure on the subject. In one embodiment, theanatomic zone of no re-flow is a disruption or obstruction of themicrovasculature of the subject. In one embodiment, the mammaliansubject is at risk for, or suffering from an anatomic zone of nore-flow. In one embodiment, the subject is at risk for, or sufferingfrom, an anatomic zone of no re-flow associated with cardiovasculartissue, or in cardiovascular tissue. In one embodiment, the subject isat risk for, or suffering from, an anatomic zone of no re-flowassociated with cerebral tissue, or in cerebral tissue. In oneembodiment, the subject is at risk for, or suffering from, an anatomiczone of no re-flow associated with renal tissue, or in renal tissue. Inone embodiment, the subject is at risk for, or suffering from, ananatomic zone of no re-flow associated with skeletal tissue, or inskeletal tissue. In one embodiment, the anatomic zone of no re-flow hasa disruption or obstruction of the microvasculature of the subject. Inone embodiment, the subject is administered the peptide prior toformation of the anatomic zone of no re-flow. In one embodiment, thesubject is administered the peptide after the formation of the anatomiczone no re-flow.

In one embodiment, the subject is administered the peptide prior to therevascularization procedure. In another embodiment, the subject isadministered the peptide after the revascularization procedure. Inanother embodiment, the subject is administered the peptide during andafter the revascularization procedure. In yet another embodiment, thesubject is administered the peptide continuously before, during, andafter the revascularization procedure.

In one embodiment, the subject is administered the peptide for at least3 hours, at least 5 hours, at least 8 hours, at least 12 hours, or atleast 24 hours after the revascularization procedure. In one embodiment,the subject is administered the peptide starting at least 8 hours, atleast 4 hours, at least 2 hours, at least 1 hour, or at least 10 minutesprior to the revascularization procedure.

In various embodiments, the subject is suffering from a myocardialinfarction, a stroke, or is in need of angioplasty. In one embodiment,the revascularization procedure is selected from the group consistingof: balloon angioplasty; insertion of a bypass graft; insertion of astent; percutaneous transluminal coronary angioplasty; or directionalcoronary atherectomy. In one embodiment, the revascularization procedureis removal of an occlusion. In one embodiment, the revascularizationprocedure is administration of one or more thrombolytic agents. In oneembodiment, the one or more thrombolytic agents are selected from thegroup consisting of: tissue plasminogen activator, urokinase;prourokinase; streptokinase; acylated form of plasminogen; acylated formof plasmin; and acylated streptokinase-plasminogen complex.

In some embodiments, the vessel occlusion is selected from the groupconsisting of: deep venous thrombosis; peripheral thrombosis; embolicthrombosis; hepatic vein thrombosis; sinus thrombosis; venousthrombosis; an occluded arterio-venal shunt; and an occluded catheterdevice.

In one aspect, the present disclosure provides a method of coronaryrevascularization comprising: (a) administering to a mammalian subject atherapeutically effective amount of the peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof such as acetate salt or trifluoroacetate salt; and (b)performing a coronary artery bypass graft procedure on the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the study design for animals used in theexamples.

FIGS. 2A and 2B are study flow charts illustrative of in vivo studyprocedure and pathologic measurements, respectively.

FIG. 3 illustrates the use of histological staining technique to assessheart tissue from a control rabbit subjected to heartischemia/reperfusion using the model described in Example 1. Panels Athrough C show exemplary heart slices from one representative controlrabbit. Panel A: Photograph showing the ischemic risk area. The coronaryartery is re-occluded at the end of the reperfusion period and Unisperseblue is injected through the left atrial catheter. Blue areas areperfused zones within the heart, and areas lacking blue dye are notperfused. Panel B: No-reflow zone (thioflavin S staining as photographedunder ultraviolet light). Thioflavin S is injected through the leftatrial catheter at the end of the reperfusion period before re-occludingthe artery. Regions with intact vessels are fluorescent, and areas ofno-reflow appear dark. Panel C: Heart slices after incubation in TTC.Regions of necrotic tissue appear white, and non-necrotic tissue stainsred.

FIG. 4 is a photograph showing an illustrative embodiment of thehemorrhagic region contained within the risk zone.

FIG. 5 is a graph showing the effect of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ onthe mean IA/AR in a rabbit model of AMI.

FIG. 6 is a graph showing the relationship between the necrotic zone andthe risk zone in the treated and control groups in a rabbit model ofAMI.

FIG. 7 is a graph showing the relationship between the risk zone and theno-reflow zone in the treated and control groups in a rabbit model ofAMI.

FIG. 8 is a graph showing the relationship between the risk zone and theno-reflow zone in the treated and control groups in a rabbit model ofAMI.

FIGS. 9A and 9B are graphs showing the infarct size and no-reflow zonesin control and treated groups in a rabbit model of AMI.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid llybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. In some embodiments, the aromatic-cationic peptide isadministered by an intracoronary route or an intra-arterial route.Administration includes self-administration and the administration byanother.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, α-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in,cardiac ischemia-reperfusion injury or one or more symptoms associatedwith cardiac ischemia-reperfusion injury. In the context of therapeuticor prophylactic applications, the amount of a composition administeredto the subject will depend on the type and severity of the disease andon the characteristics of the individual, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of disease. The skilled artisan were able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds. In the methods described herein, thearomatic-cationic peptides may be administered to a subject having oneor more signs or symptoms of a zone of no-reflow. In other embodiments,the mammal has one or more signs or symptoms of myocardial infarction,such as chest pain described as a pressure sensation, fullness, orsqueezing in the mid portion of the thorax; radiation of chest pain intothe jaw or teeth, shoulder, arm, and/or back; dyspnea or shortness ofbreath; epigastric discomfort with or without nausea and vomiting; anddiaphoresis or sweating. For example, a “therapeutically effectiveamount” of the aromatic-cationic peptides is meant levels in which thephysiological effects of anatomic zone of no re-flow injury are, at aminimum, ameliorated.

As used herein the term “ischemia reperfusion injury” refers to thedamage caused first by restriction of the blood supply to a tissuefollowed by a sudden resupply of blood and the attendant generation offree radicals. Ischemia is a decrease in the blood supply to the tissueand is followed by reperfusion, a sudden perfusion of oxygen into thedeprived tissue.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide”, “peptide”, and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for no-reflow injury if, after receiving a therapeutic amountof the aromatic-cationic peptides according to the methods describedherein, the subject shows observable and/or measurable reduction in thesize of an anatomic zone of no-reflow. It is also to be appreciated thatthe various modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial”, which includes total butalso less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. As used herein, preventingischemia-reperfusion injury includes preventing oxidative damage orpreventing mitochondrial permeability transitioning, thereby preventingor ameliorating the harmful effects of the loss and subsequentrestoration of blood flow to the heart.

Methods of Prevention or Treatment of No-Reflow Injury

The rapid restoration of coronary flow to the jeopardized myocardium isan important part of therapy after acute myocardial infarction. Despitean open infarct-related artery, breakdown of or obstruction to coronarymicrovasculature can markedly reduce blood flow to the infarct zone,leading to the formation of an anatomic zone of no re-flow. This effectis known as the no-reflow phenomenon. The no-reflow phenomenon occurs ina notable proportion of patients with AMI, despite aggressivereperfusion therapy, and is associated with a poor prognosis. Ito, H.,No-reflow phenomenon and prognosis in patients with acute myocardialinfarction, Nature Clinical Practice Cardiovascular Medicine (2006) 3,499-506. Moreover, the extent of no-reflow is a good predictor of AMIpatient outcomes. After infarction, microvascular obstruction predictsmore frequent cardiovascular complications and relates directly tolong-term prognosis in patients with AMI (infarct expansion,hospitalization, mortality, major adverse cardiac events, etc.). Wu etal., Prognostic significance of microvascular obstruction by magneticresonance imaging in patients with acute myocardial infarction.Circulation 1998, 97: 765-772; Ndrepapa et al. 5-year prognostic valueof no-reflow phenomenon after percutaneous coronary intervention inpatients with acute myocardial infarction. J Am Coll Cardiology 2010,55(21): 2383-2389; Bolognese et al. Impact of microvascular dysfunctionon left ventricular remodeling and long-term clinical outcome afterprimary coronary angioplasty for acute myocardial infarction.Circulation 2004, 109: 1121-1126; and Reffelmann et al. No-reflowphenomenon persists long-term after ischemia/reperfusion in the rat andpredicts infarct expansion. Circulation 2003, 108: 2911-2917.

The no-reflow phenomenon can occur in other organs besides the heart,such as liver, kidney, brain, skin, etc. The no-reflow concept was firstsuggested in brain ischemia. Brains of rabbits that suffered a brief 2½minutes of ischemia had normal blood flow when the ischemia wasrelieved. When the rabbits were exposed to longer ischemic periods,normal flow to brain tissues was not restored, even after relief of thevessel obstruction. Prolonged ischemia resulted in significant changesin the microvasculature that interfered with normal flow to the braincells. The existence of this phenomenon was confirmed in a variety ofanimal models of brain ischemia. It was also shown in a variety of otherorgans, including skin, skeletal muscle, and the kidney. Moreover,microcirculation alterations can modulate the organ damage induced byischemia-reperfusion injury during organ transplantation. See Majno etal. No-reflow after cerebral ischaemia. Lancet. 1967; 2: 569-570; Ameset al. Cerebral ischemia, II: the no-reflow phenomenon. Am J Pathol.1968; 52: 437-447; Cerisoli et al. Experimental cerebral “no-reflowphenomenon”: response to intracarotid injection of dexamethasone,furosemide and escina. J Neurosurg Sci. 1981; 25: 7-12; Ito et al.Transient appearance of “no-reflow” phenomenon in Mongolian gerbils.Stroke. 1980; 11: 517-521; Asano T, Sano K. Pathogenetic role ofno-reflow phenomenon in experimental subarachnoid hemorrhage in dogs. JNeurosurg. 1977; 46: 454-466; Chait et al. The effects of the perfusionof various solutions on the no-reflow phenomenon in experimental freeflaps. Plast Reconstr Surg. 1978; 61: 421-430; Allen et al.Pathophysiology and related studies of the no-reflow phenomenon inskeletal muscle. Clin Orthop. 1995; 314: 122-133; Summers W K, Jamison RL. The no-reflow phenomenon in renal ischemia. Lab Invest. 1971; 25:635-643; Johnston W H, Latta H. Glomerular mesangial and endothelialcell swelling following temporary renal ischemia and its role in theno-reflow phenomenon. Am J Pathol. 1977; 89: 153-166.

The present technology relates to the treatment or prevention ofischemia-reperfusion injury in mammals through administration oftherapeutically effective amounts of aromatic-cationic peptides such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt to subjects inneed thereof. In one aspect, the present technology relates to methoduseful in the treatment or prevention of anatomic zone of no-reflow,zone of hemorrhage and infarct size following ischemia/reperfusion. Inone embodiment, the treatment of an anatomic zone of no-reflow includesincreasing the amount or area of tissue perfusion in a subject comparedto a similar subject not administered the aromatic-cationic peptide. Inone embodiment, the prevention of an anatomic zone of no-reflow includesreducing the amount or area of microvascular damage caused byreperfusion in a subject compared to a similar subject not administeredthe aromatic-cationic peptide. In some embodiments, treatment orprevention of an anatomic zone of no-reflow includes reducing injury tothe affected vessel upon reperfusion, reducing the effect of plugging byblood cells, and/or reducing endothelial cell swelling in a subjectcompared to a similar subject not administered the aromatic-cationicpeptide. The extent of the prevention or treatment can be measured byany technique known in the art, including but not limited to, MRI inorder to assess microvascular damage). Re-flow phenomenon may also beassessed using myocardial contrast echocardiography, coronaryangiography, myocardial blush, coronary doppler imaging,electrocardiography, nuclear imaging single-photon emission CT, usingthallium or technetium-99m, and PET. Successful prevention or treatmentcan be determined by comparing the extent of no-reflow in the subjectobserved by any of these imaging techniques compared to a controlsubject or a population of control subjects that are not administeredthe aromatic-cationic peptide.

In one aspect, the present technology relates to the treatment orprevention of an anatomic zone of no re-flow by administration ofcertain aromatic-cationic peptides, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂,or pharmaceutically acceptable salts thereof, such as acetate salt ortrifluoroacetate salt, to a subject in need thereof. In one embodiment,the administration of the aromatic-cationic peptide(s) to a subject isbefore the formation of the anatomic zone of no re-flow. In anotherembodiment, the administration of the aromatic-cationic peptide(s) to asubject is after the formation of an anatomic zone of no re-flow. In oneembodiment, the method is performed in conjunction with arevascularization procedure. Also provided is a method for the treatmentor prevention of cardiac ischemia-reperfusion injury. Also provided is amethod of treating a myocardial infarction in a subject to preventinjury to the heart upon reperfusion. In one aspect, the presenttechnology relates to a method of coronary revascularization comprisingadministering to a mammalian subject a therapeutically effective amountof the aromatic cationic peptide and performing coronary artery bypassgraft (CABG) procedure on the subject.

In one embodiment, the subject is administered the peptide such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt, prior to arevascularization procedure. In another embodiment, the subject isadministered the peptide after the revascularization procedure. Inanother embodiment, the subject is administered the peptide during andafter the revascularization procedure. In yet another embodiment, thesubject is administered the peptide continuously before, during, andafter the revascularization procedure. In another embodiment, thesubject is administered the peptide regularly (i.e., chronically)following an AMI and/or a revascularization or CABG procedure.

In some embodiments, the subject is administered the peptide after therevascularization procedure. In one embodiment, the subject isadministered the peptide for at least 3 hours, at least 5 hours, atleast 8 hours, at least 12 hours, or at least 24 hours after therevascularization procedure. In some embodiments, the subject isadministered the peptide prior to the revascularization procedure. Inone embodiment, the subject is administered the peptide starting atleast 8 hours, at least 4 hours, at least 2 hours, at least 1 hour, orat least 10 minutes prior to the revascularization procedure. In oneembodiment, the subject is administered for at least one week, at leastone month or at least one year after the revascularization procedure. Insome embodiments, the subject is administered the peptide prior to andafter the revascularization procedure. In some embodiments, the subjectis administered the peptide as an infusion over a specified period oftime. In some embodiments, the peptide is administered to the subject asa bolus.

The aromatic-cationic peptides are water-soluble and highly polar.Despite these properties, the peptides can readily penetrate cellmembranes. The aromatic-cationic peptides typically include a minimum ofthree amino acids or a minimum of four amino acids, covalently joined bypeptide bonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the a positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alaninc (Ala), argininc (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. In some embodiments, the peptide has no amino acids thatare naturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include β-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. In some embodiments, the peptidehas only D-amino acids, and no L-amino acids. If the peptide containsprotease sensitive sequences of amino acids, at least one of the aminoacids is preferably a non-naturally-occurring D-amino acid, therebyconferring protease resistance. An example of a protease sensitivesequence includes two or more contiguous basic amino acids that arereadily cleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH were referred to below as (p_(m)).The total number of amino acid residues in the peptide were referred tobelow as (r). The minimum number of net positive charges discussed beloware all at physiological pH. The term “physiological pH” as used hereinrefers to the normal pH in the cells of the tissues and organs of themammalian body. For instance, the physiological pH of a human isnormally approximately 7.4, but normal physiological pH in mammals maybe any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-argininc, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-D-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negativelycharged amino acid (i.e., Glu) and four positively charged amino acids(i.e., two Arg residues, one Lys, and one His). Therefore, the abovepeptide has a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1.

In this embodiment, the relationship between the minimum number of netpositive charges (p_(m)) and the total number of amino acid residues (r)is as follows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups werereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (P_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

2′,6′-Dmp-D-Arg-2′,6′-Dmt-Lys-NH₂ 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂2′,6′-Dmt-D-Arg-PheOrn-NH₂2′,6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH₂2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Cit-PheLys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D- Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′,6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂D-Tyr-Trp-Lys-NH₂Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met- NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂ Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂ Met-Tyr-D-Arg-Phe-Arg-NH₂Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-Asp Phe-D-Arg-2′,6′-Dmt-Lys-NH₂Phe-D-Arg-His Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH₂Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂ Tyr-D-Arg-Phe-Lys-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Mu-opioid activity can beassessed by radioligand binding to cloned mu-opioid receptors or bybioassays using the guinea pig ileum (Schiller et al., Eur J Med Chem,35:895-901, 2000; Zhao et al., J Pharmacol Exp Ther, 307:947-954, 2003).Activation of the mu-opioid receptor typically elicits an analgesiceffect. In certain instances, an aromatic-cationic peptide havingmu-opioid receptor agonist activity is preferred. For example, duringshort-term treatment, such as in an acute disease or condition, it maybe beneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. Such acute diseases and conditions are oftenassociated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic-cationic peptide may be beneficial inthe treatment regimen of the human patient or other mammal. Anaromatic-cationic peptide which does not activate the mu-opioidreceptor, however, may also be used with or without an analgesic,according to clinical requirements.

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor agonist activity is preferred. Forexample, during long-term treatment, such as in a chronic disease stateor condition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances, thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatient or other mammal. Potential adverse effects may include sedation,constipation and respiratory depression. In such instances anaromatic-cationic peptide that does not activate the mu-opioid receptormay be an appropriate treatment.

Peptides which have mu-opioid receptor agonist activity are typicallythose peptides which have a tyrosine residue or a tyrosine derivative atthe N-terminus (i.e., the first amino acid position). Suitablederivatives of tyrosine include 2′-methyltyrosine (Mmt);2′,6′-dimethyltyrosine (2′,6′-Dmt); 3′,5′-dimethyltyrosine (3′5′Dmt);N,2′,6′-trimethyltyrosine (Tmt); and 2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂. This peptide has a netpositive charge of three, contributed by the amino acids tyrosine,arginine, and lysine and has two aromatic groups contributed by theamino acids phenylalanine and tyrosine. The tyrosine can be a modifiedderivative of tyrosine such as in 2′,6′-dimethyltyrosine to produce thecompound having the formula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂. This peptidehas a molecular weight of 640 and carries a net three positive charge atphysiological pH. The peptide readily penetrates the plasma membrane ofseveral mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther., 304:425-432, 2003).

Peptides that do not have mu-opioid receptor agonist activity generallydo not have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂.Alternatively, the N-terminal phenylalanine can be a derivative ofphenylalanine such as 2′,6′-dimethylphenylalanine (2′,6′-Dmp). In oneembodiment, a peptide with 2′,6′-dimethylphenylalanine at amino acidposition 1 has the formula 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In oneembodiment, the amino acid sequence is rearranged such that Dmt is notat the N-terminus. An example of such an aromatic-cationic peptide thatdoes not have mu-opioid receptor agonist activity has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

The peptides mentioned herein and their derivatives can further includefunctional analogs. A peptide is considered a functional analog if theanalog has the same function as the stated peptide. The analog may, forexample, be a substitution variant of a peptide, wherein one or moreamino acids are substituted by another amino acid. Suitable substitutionvariants of the peptides include conservative amino acid substitutions.Amino acids may be grouped according to their physicochemicalcharacteristics as follows:

(a) Non-polar amino acids: Ala (A) Ser (S) Thr (T) Pro (P) Gly (G) Cys(C);

(b) Acidic amino acids: Asn (N) Asp (D) Glu (E) Gln (Q);

(c) Basic amino acids: His (H) Arg (R) Lys (K);

(d) Hydrophobic amino acids: Met (M) Leu (L) Ile (I) Val (V); and

(e) Aromatic amino acids: Phe (F) Tyr (Y) Trp (W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 5.

TABLE 5 Peptide Analogs with Mu-Opioid Activity Amino Amino Amino AcidAcid Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH- NH₂ dns 2′6′Dmt D-Arg PheLys-NH(CH₂)₂—NH- NH₂ atn 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit PheLys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic NH₂ acid) Bio- D-Arg Phe Lys NH₂ 2′6′Dmt 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg TyrDap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of analogs that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs Lacking Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc, New York (1997).

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

General.

The aromatic-cationic peptides described herein, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt, are useful toprevent or treat a disease or condition. Specifically, the disclosureprovides for both prophylactic and therapeutic methods of treating asubject at risk of (or susceptible to) vessel occlusion injury, ananatomic zone of no re-flow or cardiac ischemia-reperfusion injury.Accordingly, the present methods provide for the prevention and/ortreatment of vessel occlusion injury, cardiac ischemia-reperfusioninjury or an anatomic zone of no-reflow in a subject by administering aneffective amount of an aromatic-cationic peptide to a subject in needthereof.

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment. In various embodiments, in vitro assays can be performedwith representative animal models, to determine if a givenaromatic-cationic peptide-based therapeutic exerts the desired effect inpreventing or treating an anatomic zone of no re-flow. Compounds for usein therapy can be tested in suitable animal model systems including, butnot limited to rats, mice, chicken, pigs, cows, monkeys, rabbits, sheep,guinea pig, and the like, prior to testing in human subjects. Similarly,for in vivo testing, any of the animal model systems known in the artcan be used prior to administration to human subjects.

Prophylactic Methods.

In one aspect, the invention provides a method for preventing, in asubject an anatomic zone of no re-flow by administering to the subjectan aromatic-cationic peptide that prevents the initiation or progressionof the condition. Subjects at risk for an anatomic zone of no re-flowcan be identified by, e.g., any or a combination of diagnostic orprognostic assays as described herein. In prophylactic applications,pharmaceutical compositions or medicaments of aromatic-cationic peptidesare administered to a subject susceptible to, or otherwise at risk of adisease or condition in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease orcondition, including biochemical, histologic and/or behavioral symptomsof the disease or condition, its complications and intermediatepathological phenotypes presenting during development of the disease orcondition. Administration of a prophylactic aromatic-cationic can occurprior to the manifestation of symptoms characteristic of the aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. The appropriate compound can be determined based onscreening assays described above.

Therapeutic Methods.

Another aspect of the technology includes methods of treating vesselocclusion injury, an anatomic zone of no re-flow or cardiacischemia-reperfusion injury in a subject for therapeutic purposes. Intherapeutic applications, compositions or medicaments are administeredto a subject suspected of, or already suffering from such a disease orcondition in an amount sufficient to cure, or at least partially arrest,the symptoms of the disease or condition, including its complicationsand intermediate pathological phenotypes in development of the disease.or condition As such, the invention provides methods of treating anindividual afflicted with an anatomic zone of no re-flow.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, suitably a human. When used in vivo fortherapy, the aromatic-cationic peptides are administered to the subjectin effective amounts (i.e., amounts that have desired therapeuticeffect). The dose and dosage regimen will depend upon the degree of theinjury in the subject, the characteristics of the particulararomatic-cationic peptide used, e.g., its therapeutic index, thesubject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods may be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide may be administeredsystemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphorsulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the salt is an acetate salt or atrifluoroacetate salt.

The aromatic-cationic peptides described herein, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt, can beincorporated into pharmaceutical compositions for administration, singlyor in combination, to a subject for the treatment or prevention of adisorder described herein. Such compositions typically include theactive agent and a pharmaceutically acceptable carrier. As used hereinthe term “pharmaceutically acceptable carrier” includes saline,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomerasol, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, itwere preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Preferably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.1-10,000 micrograms per kg body weight. In one embodiment,aromatic-cationic peptide concentrations in a carrier range from 0.2 to2000 micrograms per delivered milliliter. An exemplary treatment regimeentails administration once per day or once a week. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹² to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.01 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.01 to about 0.5 mg/kg/h, suitably fromabout 0.0001 to about 0.1 mg/kg/h. In one embodiment, the mid-dose isprovided from about 0.001 to about 1.0 mg/kg/h, suitably from about 0.01to about 0.5 mg/kg/h. In one embodiment, the high dose is provided fromabout 0.005 to about 10 mg/kg/h, suitably from about 0.01 to about 2mg/kg/h.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a suitable embodiment, the mammal is a human.

Measurement of the Anatomic Zone of No-Reflow

Imaging techniques are useful in assessing the effect of the peptides ofthe present technology on the anatomic zone of no re-flow. (Seegenerally reference nos. 1-51). Re-flow phenomenon may be assessed usingmyocardial contrast echocardiography, coronary angiography, myocardialblush, coronary doppler imaging, electrocardiograph, nuclear imagingsingle-photon emission CT, using thallium or technetium-99m, and PET.Contrast-enhanced MRI can evaluate myocardial perfusion during firstpass of the contrast agent. Alternatively, delayed contrast-enhanced MRI20 min after contrast injection can be used to detect necrosis. Thedetection of hypoenhancement zones on first-pass perfusion MRI, whichrepresents no re-flow, is associated with permanent dysfunction atfollow-up. In some embodiments, microvascular obstructions can beassessed using cardiac MRI. For example, a 1.5-T body MRI scanner can beused to perform cardiac MRI in order to assess ventricular function,myocardial edema (area at risk), microvascular obstruction and infarctsize.

EXAMPLES

The present invention is further illustrated by the following example,which should not be construed as limiting in any way.

As noted above, ischemia can result in significant changes in themicrovasculature that interferes with normal blood flow to manytissues/organs. As such, the no-reflow phenomenon can occur in a varietyof tissues/organs including heart, liver, brain, skin, skeletal muscle,kidney, etc. It is predicted that the aromatic-cationic peptides of thepresent technology are useful methods to prevent or treat the zone ofanatomic no-reflow in a variety of tissues/organs.

Example 1. Effects of D-Ara-2′,6′-Dmt-Lys-Phe-NH₂ on the Anatomic Zoneof No-Reflow, Zone of Hemorrhage and Infarct Size FollowingIschemia/Reperfusion Insult in the Rabbit Heart

General.

The effects of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on infarct size and no-reflowafter regional myocardial ischemia (approximately 30 min) andreperfusion (approximately 3 h) are studied. The efficacy of the studyarticles (i.e., vehicle/placebo and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetatesalt)) are assessed with experimental procedures that differ by the timepoint of study article administration relative to the onset of ischemiaand subsequent myocardial reperfusion.

Route and Duration of Administration.

The vehicle/placebo and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) areadministered via intravenous infusion. Details regarding infusion andduration of infusion are provided in FIG. 1.

Test Animals.

Animals utilized in the studies are detailed in Table 7. Animals aremaintained in animal facility in accordance with standards set by theUSDA and NIH. Prior to randomization into the Protocol, rabbits areexamined by qualified personnel and acclimated for a period of at least2 days. All rabbits are observed daily for signs of diarrhea, lack ofappetite, malaise and the like. If any of these signs are seen, therabbit is not included in the study population. Animals are fasted(removal of food) overnight immediately prior to that animal'sexperiment. All animals are absent of obvious clinical disease. Bodyweights are recorded for each rabbit either the day before that animal'sexperiment or the day of that animal's experiment for the purpose ofdose formulation calculation and anesthesia. Following the completion ofeach experiment, animals (already fully anesthetized) are euthanatizedand disposed of appropriately.

TABLE 7 Description of Test Animals Species Rabbits Strain New ZealandWhite Sex Male Number of animals Approximately 30-50 Age at start ofstudy Approximately 4-7 months Weight at start of study Approximately2.0-3.75 kilograms

Study Articles (D-Arg-2′,6′-Dmt-Lys-Phe-N_(H)(Acetate Salt) andVehicle/Placebo).

The lot number, supplier name, peptide content, expiration date, andstorage condition of the study articles described below are recorded.The study article formulations for D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetatesalt) are prepared as weight-to-volume mixtures in vehicle as detailedbelow.

D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) (sterile lyophilized powder)used in these studies is supplied by Stealth Peptides, Inc.D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) study article is dissolved inthe sterile normal saline (0.9% NaCl) to make anD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) study article stock solution.After reconstitution with saline, the D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) study article stock solution is filtered using a sterilefilter unit with a membrane of less than 0.22 μm (a PES or PVDFmembrane) and stored at −20° C. until use.

On the morning of each D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt)experiment, the aliquoted frozen D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetatesalt) study article stock solution is thawed at room temperature beforemixing it with sterile normal saline (0.9% NaCl). The vehicle/placebo issterile normal saline (0.9% NaCl). The concentration/dose ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) to be used in the studies issummarized in Tables 8-11 for dosing solution preparation and infusionrates.

TABLE 8 Dosing Preparation for D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ Test articleD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) Vehicle Sterile saline (0.9%NaCl) Study Group Group 1: 0.05 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt), infusion for the entire 200 minutes (20′ ischemia + 180′reperfusion) Group 2: 0.075 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt), infusion for the first 20 minutes, then change to 0.05mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt), infusion for 170minutes. Total infusion time = 190 minutes (10′ ischemia + 180 minreperfusion) Group 3: 0.10 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt), infusion for the first 20 minutes, then switch to 0.05mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt), infusion for 160minutes. Total infusion time is equal to 180 minutes (180 minreperfusion only)

TABLE 9 Dosing Preparation for D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ For Group 1:0.05 mg/kg/hr infusion for 200 minutes Peptide Total Dose conc. BodyInfusion rate infusion Infusion (mg/kg/ (mg/ weight (mL/ (mL/ volumeschedule hr) mL) (kg) min) hr) (mL) From 0′ 0.050 0.10 2.0 0.017 1.003.3 to 200′ 0.050 0.10 2.5 0.021 1.25 4.2 0.050 0.10 3.0 0.025 1.50 5.00.050 0.10 3.5 0.029 1.75 5.8 0.050 0.10 4.0 0.033 2.00 6.7

TABLE 10 Dosing Preparation for D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ For Group 2:0.075 mg/kg/hr infusion for 20 minutes; switch to 0.05 mg/kg/hr for 170minutes. Total infusion time = 190 minutes Peptide Total Dose Conc. BodyInfusion rate Infusion infusion Infusion (mg/kg/ (mg/ weight (mL/ (mL/volume volume Schedule hr) mL) (kg) min) hr) (mL) (mL) From 0′ 0.0750.10 2.0 0.025 1.50 0.50 3.3 to 20′ From 21′ 0.050 0.10 2.0 0.017 1.002.83 to 190′ From 0′ 0.075 0.10 2.5 0.031 1.88 0.63 4.2 to 20′ From 21′0.050 0.10 2.5 0.021 1.25 3.54 to 190′ From 0′ 0.075 0.10 3.0 0.038 2.250.75 5.0 to 20′ From 21′ 0.050 0.10 3.0 0.025 1.50 4.25 to 190′ From 0′0.075 0.10 3.5 0.044 2.63 0.88 5.8 to 20′ From 21′ 0.050 0.10 3.5 0.0291.75 4.96 to 190′ From 0′ 0.075 0.10 4.0 0.050 3.00 1.00 6.7 to 20′ From21′ 0.050 0.10 4.0 0.033 2.00 5.67 to 190′

TABLE 11 Dosing Preparation for D-Arg-2′,6′-Dmt-Lys-Phe- NH₂ For Group3: 0.10 mg/kg/hr infusion for 20 minutes; switch to 0.05 mg/kg/hr for160 minutes. Total infusion time = 180 minutes (180′ reperfusion only)Peptide Total Dose Conc. Body Infusion rate Infusion infusion Infusion(mg/kg/ (mg/ weight (mL/ (mL/ volume volume Schedule hr) mL) (kg) min)hr) (mL) (mL) From 0′ 0.100 0.10 2.0 0.033 2.00 0.67 3.3 to 20′ From 21′0.050 0.10 2.0 0.017 1.00 2.67 to 190′ From 0′ 0.100 0.10 2.5 0.042 2.500.83 4.2 to 20′ From 21′ 0.050 0.10 2.5 0.021 1.25 3.33 to 190′ From 0′0.100 0.10 3.0 0.050 3.00 1.00 5.0 to 20′ From 21′ 0.050 0.10 3.0 0.0251.50 4.00 to 190′ From 0′ 0.100 0.10 3.5 0.058 3.50 1.17 5.8 to 20′ From21′ 0.050 0.10 3.5 0.029 1.75 4.67 to 190′ From 0′ 0.100 0.10 4.0 0.0674.00 1.33 6.7 to 20′ From 21′ 0.050 0.10 4.0 0.033 2.00 5.33 to 190′

Study Design.

Studies are designed utilizing an adaptive design model. As such, thedata collected in the first group of approximately 32 rabbits (Cohort 1)is used to modify, where appropriate, the remainder of the studies(e.g., subsequent cohorts). After approximately 15-30 min ofstabilization, baseline hemodynamic parameters and temperatures areobtained. Furthermore, the rabbits are randomized to one of thefollowing four groups (see FIG. 1): Group 1, Study articleD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt): D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) infusion starting at approximately 10 minutes aftercoronary artery occlusion (CAO) and continuing throughout approximately180 minutes of reperfusion, n=8 (dose/volume to be determined);D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) infusion time is equal toapproximately 200 minutes; Group 2, Study articleD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt): D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) infusion starting approximately 20 minutes after CAO, (10minutes before reperfusion) and continuing throughout approximately 180minutes of reperfusion, n=8 (dose/volume to be determined);D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) infusion time is equal toapproximately 190 minutes; Group 3, Study articleD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt): D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) starting immediately prior to coronary artery reperfusionand continuing throughout approximately 180 minutes of reperfusion, n=8(dose/volume to be determined); D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetatesalt) infusion time is equal to approximately 180 minutes; and Group 4,Study article (control): An equivalent volume relative toD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) of vehicle/placebo infusionstarting at approximately 10 minutes after CAO and continuing throughoutapproximately 180 minutes of reperfusion, n=8. Vehicle/placebo infusiontime is equal to approximately 200 minutes.

To achieve desired plasma concentrations withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ for specific time points (e.g., uponreperfusion, 15 minutes after reperfusion begins and approximatelysteady state), the dosing design for each of Groups 1, 2 and 3 should beas follows:

-   -   Group 1, 0.05 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate        salt), infusion for the entire 200 minutes of Group 1,    -   Group 2, 0.075 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate        salt), infusion for the first 20 minutes, then change to 0.05        mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt), infusion        for 170 minutes of Group 2, and    -   Group 3, 0.10 mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate        salt), infusion for the first 20 minutes, then change to 0.05        mg/kg/hr of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt), infusion        for 160 minutes of Group 3.

Surgical Preparation (See FIG. 2A: Study Flow Chart—In-Vivo Proceduresand FIG. 2B: Study Flow Chart—Pathologic Measurements).

Male New Zealand White rabbits are anesthetized with an intramuscularinjection of a mixture of ketamine (approximately 75 mg/kg) and xylazine(approximately 5 mg/kg). Pentobarbital anesthesia are givenintravenously during the study as required to maintain a deep level ofanesthesia. The rabbits are intubated and mechanically ventilated withoxygen-enriched air. Fluid-filled catheters are inserted into the leftjugular vein to administer the study articles, into the right jugularvein to administer anesthesia, and into the left carotid artery toassess hemodynamics and to take a reference blood sample during regionalmyocardial blood flow measurement. The chest are opened through the leftfourth intercostal space, the pericardium incised, and the heartexposed. Near the base of the heart, the first large antero-lateralbranch of the circumflex artery, or the circumflex artery itself, areencircled with a 4-O silk suture. CAO in this region normally results inischemia of a large territory of the antero-lateral and apicalventricular wall. The ends of the suture are threaded through a piece oftubing, forming a snare that are tightened to occlude the artery. Acatheter is placed into the left atrial appendage to inject radioactivemicrospheres, and dyes at the end of the study. A temperature probe isinserted into the rectum, and body temperature is maintained using aheating pad. All rabbits will undergo approximately 30 minutes of CAOand reperfusion for approximately 3 hours. Regional myocardial bloodflow, using the radioactive microsphere technique, are measured atapproximately 20-30 minutes of CAO to ensure ischemia in the risk zoneand that the degree of ischemia are equivalent in the four groups.

Route and Duration of Study Articles.

Pump infusion rate and volume are calibrated before initiation of thestudy. The study articles are administered via the jugular catheterusing an infusion pump at rates to be determined. Infusion at the timeappropriate for the particular group and continue throughoutapproximately 180 minutes of reperfusion.

Data Procedures, Collection, and Measurements.

Heart rate, systolic and diastolic pressures, and rectal temperature aremonitored and recorded at approximately baseline, 5, 15 and 29 minutesof occlusion, and at approximately 5, 15, 30, 60, 90, 120, 150 and 180minutes of reperfusion. These times points may vary slightly to accountfor the stability or instability of the animal.

At approximately 180 minutes, approximately 1 ml/kg of a ˜4% solution ofthioflavin S is injected into the heart via the left atrial catheter todefine the region of no-reflow. Thioflavin S, a fluorescent green-yellowdye, stains endothelium, and serves as a marker of perfusion. ThioflavinS is used as a standard marker for identifying zones of no-reflow (SeeFIG. 3). The coronary artery is re-occluded and the ischemic risk regiondelineated with approximately 4 ml of a ˜50% solution of Unisperse bluedye (supplier Ciba Geigy) injected into the left atrium.

Regional Myocardial Blood Flow Technique (RMBF).

RMBF are measured at approximately 25 minutes of ischemia usingapproximately 2×10⁶ radioactive microspheres (supplier PerkinElmer LifeSciences). The microspheres are injected into the left atrium throughthe left atrial catheter, and a reference blood sample are obtained fromthe carotid artery at the rate of approximately 2.06 ml/min. At the endof the Protocol, samples are cut from the risk region (determined by theabsence of blue dye) and from the non-ischemic region. The samples areweighed and counted together with the reference blood samples in acomputerized gamma well counter (supplier Canberra, System S100). Afterappropriate subtraction of backgrounds and correction for overlappingradioactivity between isotopes, RMBF are computed and the results areexpressed as ml/min/g (See FIG. 3).

Hemodynamic Measurements.

Heart rate and blood pressures are measured using a fluid filledcatheter inserted into a carotid artery. Data are digitized and recordedusing an ADI (supplier Advanced Digital Instruments) system. Three beatsare averaged for each time point.

Analysis of No-Reflow, Risk and Hemorrhagic Zones, and Necrosis.

The heart are sliced transversely into 6-8 sections. The slices arephotographed under ultraviolet light to identify the region of no-reflow(see FIG. 3, panel B) and under halogen lighting to identify the area atrisk (see FIG. 3, panel A) and the hemorrhagic area (see FIG. 4). Theslices are then be incubated in a ˜1% solution of triphenyltetrazoliumchloride (TTC) for approximately 15 minutes, immersed in formalin, andre-photographed in order to visualize the infarcts (see FIG. 3, panelC). Necrotic tissue fails to stain with TTC and appears white to paleyellow. Non-infarcted tissue stains brick red. The digital photographsare observed on a computer monitor. The areas of no-reflow, hemorrhage,ischemic and normally perfused regions, and the areas of necrotic andnon-necrotic regions in each slice are digitized using Image J (supplierRasband W S, Image J, National Institutes of Health,http://rsb.info.nih.gov/ij/). The areas in each slice are multiplied bythe weight of the slice and the results will summed in order to obtainthe mass of the no-reflow, risk and infarcted areas.

One slice from the center of the infarcted region containing necroticand normal zones are examined by histology and any edema are scoredsemi-quantitatively (scale of 0-3+). All photographic measurements areperformed by a trained individual that is blinded to the study protocol.

Sample Size, Exclusion Criteria and Data Analyses.

Initial sample sizes will consist of n=8 animals in each group.Exclusion criteria include: death occurring before reperfusion and thestudy is completed, or an ischemic risk zone of <10% of the leftventricle. Data analyses include excluding animals in each group with aregional myocardial blood flow >0.2 ml/min/g within the risk zone duringCAO, and inclusion of animals in each group with a regional myocardialblood flow >0.2 ml/min/g within the risk zone during CAO.

Blood Samples Collection and Handling.

Venous whole blood samples are collected for pharmacokinetic (PK)analyses. Table 12 summarizes collection time points. Venous whole bloodsamples are collected for PK analyses at the time points specifiedbelow. For PK analysis samples 1 ml venous whole blood is collectedusing pre-chilled syringe into the pre-chilled BD Vacutainer® PlasmaSeparation Tubes (lavender top) containing (spray coated) K₂EDTA at thefollowing time points: for Group 1, 2 and 4, approximately baseline, 29to 30 mins of ischemia (before on-set of the reperfusion). andapproximately 30, 60 and 180 mins post-reperfusion. For Group 3,approximately 15, 30, 90 and 180 mins post-reperfusion.

TABLE 12 Summary of Collection Time Points Surgery Ischemia Reperfusionprep and (30 mins) (180 mins) stabilization 0 5 15 29 to 30 5 15 30 6090 120 150 180 For Group 1, 2 and 4, x x x x x venous blood draw for PKanalysis (~1 ml plasma collection) For Group 3, x x x x x venous blooddraw for PK analysis (~1 ml plasma collection) Temperature* x x x x x xx x x x x x HR* x x x x x x x x x x x x Hemodynamic data* x x x x x x xx x x x x (ABP)

The PK blood tubes is adequately and gently mixed and placed immediatelyinto ice.

Within 30 minutes of collection, samples are centrifuged atapproximately 1500 up to, preferably, 2000×G for 15 min at ˜4° C.,following which two plasma aliquots (approximately 0.25 ml each) areremoved and immediately placed into labelled screw-cap polypropylenetubes. Individual plasma samples are quick frozen over dry ice, storedfrozen under conditions set to maintain ˜70° C.±15° C., until furtheranalysis.

Statistical Analysis Plan.

All data summaries and statistical analyses are performed using SAS(Version 9.3). Left ventricular weight, infarct size, area at risk, areaof no-reflow and hemorrhage and blood flow are compared using analysisof variance. The differences among groups are to be determined byTukey's test. Changes in hemodynamic variables over time are analyzed byanalysis of variance (repeated measures). If an f-value of <0.05 isobtained for the animal model, differences among means are determined bythe method of contrasts. Analysis of covariance (ANCOVA) will also beused to test for a group effect on the regression models of necroticmyocardium with risk, no-reflow, and hemorrhage zones, and collateralblood flow. Data are expressed as mean±SEM.

End Points.

Endpoint for the studies may include, but are not limited to, heartrate, systolic/diastolic arterial pressure, pathologic specimen areasand their respective ratios, area at risk zone, infarct mass, leftventricular mass, risk zone, no-reflow zone, hemorrhage zone,necrosis/infarct size, and edema.

Other aromatic-cationic peptides, for example, Phe-D-Arg-Phe-Lys-NH₂ orpharmaceutically acceptable salts thereof, such as acetate salt ortrifluoroacetate salt, could be tested in a similar manner.

Example 2. Effect of D-Arg-2′,6′-Dmt-Lys-Phe-NH_on the Mean IA/AR in aRabbit Model of AMI

Studies were conducted to assess the effect ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on mean IA/AR in a rabbit model of AMI asdescribed in Example 1. The effect of the timing of the infusion ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) was assessed. The effect ofthe timing of the infusion of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt)at a low dose (from about 0.01 to about 0.5 mg/kg/h) was assessed. Theresults are shown in Table 13 and FIG. 5. D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ isuseful in the treatment of ischemic injury as well as the prevention andtreatment of reperfusion injury in heart as judged by a reduction inIA/AR ratio in subjects administered D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) compared with subjects receiving placebo. In someexperiments, the infarct size was reduced an average of 11% (not shown).In some experiments, the infarct size was reduced by up to 44.7% (Table13; FIG. 5).

TABLE 13 Effect of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on Mean IA/AR in a RabbitModel of AMI Treatment Mean IA/AR Results Placebo 37.4% NAD-Arg-2′,6′-Dmt-Lys-Phe- 20.7% ↓44.7% from placebo NH₂ (acetate salt)(started 10 min into 30 min of ischemia D-Arg-2′,6′-Dmt-Lys-Phe- 27.9%↓25.5% from placebo NH₂ (acetate salt) (started 20 min into 30 min ofischemia D-Arg-2′,6′-Dmt-Lys-Phe- 26.7% ↓25.5% from placebo NH₂ (acetatesalt) (started 30 min into ischemia immediately prior to reperfusion)

Other aromatic-cationic peptides, for example, Phe-D-Arg-Phe-Lys-NH₂ orpharmaceutically acceptable salts thereof, such as acetate salt ortrifluoroacetate salt, could be tested in a similar manner. It ispredicted that similar results would be achieved with the alternativepeptides.

Example 3. Effects of D-Ara-2′,6′-Dmt-Lys-Phe-NH₂ on the Anatomic Zoneof No-Reflow, Zone of Hemorrhage and Infarct Size FollowingIschemia/Reperfusion Insult in the Rabbit Heart

Studies were conducted to assess the effect ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on the anatomic zone of no-reflow, zone ofhemorrhage and infarct size in a rabbit model of AMI as described inExample 1. The results show that D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ is usefulin the treatment of ischemic injury as well as the prevention andtreatment of reperfusion injury in heart, including an anatomical nore-flow zone.

Sixty-six rabbits entered the protocol which are inclusive of theanimals detailed in Example 2. Data from two hearts were excluded basedon the prospective exclusion criterion of ischemic risk zone size(AR<10% of the left ventricle). Final data are reported on the remaining64 hearts: Group 1, n=15 (D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt)infusion starting at 10′ after coronary artery occlusion [CAO]; Group 2,n=17 (D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) infusion starting at20′ after CAO); Group 3, n=17 (D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetatesalt) infusion starting at 29′ after CAO); Group 4, n=15 (salineinfusion starting at 10′ after CAO). Three hearts had RMBF >0.20ml/min/g in the ischemic zone during CAO; 3 rabbits may have hadelevated serum levels of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ due to issues withthe infusion pump. These hearts were included in the data analysespresented here.

Ischemic risk zone, no-reflow zone, hemorrhagic zone and infarct size inthe 4 groups is shown in Table 14. Mean and standard error values forthese variables in the 4 group are provided. Potential differences amongthese variable were analyzed by analysis of variance.

TABLE 14 All hearts with AR >10% of the left ventricle Group 1 Group 2Group 3 N = 15 N = 17 N = 17 D-Arg-2′,6′- D-Arg-2′,6′-Dmt-D-Arg-2′,6′-Dmt- Dmt-Lys-Phe- Lys-Phe-NH₂ Lys-Phe-NH₂ Group 4 NH₂(acetate (acetate salt) 20′ (acetate salt) 29′ N = 15 salt) 10′ occloccl occl saline Area at risk/left ventricle 0.30 ± 0.02 0.32 ± 0.020.31 ± 0.02 0.30 ± 0.02 No-reflow/area at risk 0.23 ± 0.02 0.21 ± 0.030.21 ± 0.03 0.28 ± 0.03 No-reflow/area of 0.74 ± 0.04 0.63 ± 0.05 0.70 ±0.09 0.80 ± 0.05 necrosis Occl = coronary artery occlusion

Analysis of Covariance was used to test the class variable “group” foran effect on the relationship between the extent of the risk region(AR/LV) expressed in grams and the extent of necrosis (AN/LV) (FIG. 6)and the extent of the risk region and that of the no-reflow region (FIG.7). There was a significant effect of group on the relationship betweenno-reflow and risk. The regression lines for the treated groups liebelow that of the control group. An analysis of the necrotic areadivided by area-at-risk (AN/AR) for rabbits with AR/LV >15%, showed thattreatment with D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ results in a statisticallysignificant reduction of infarct size of greater than 20% (See Table15).

TABLE 15 Necrotic Area Divided By Area-At-Risk For Rabbits WithAR/LV >15% FOR ALL ACTIVE GROUPS COMBINED SUM 15.245 N 48 MEAN 0.318REDUCTION IN AN/AR WITH D-Arg-2′,6′-Dmt- 20.2% Lys-Phe-NH₂ C/W PLACEBO

ANCOVA testing for group effect was performed on the combined groupdata. There was a significant group effect on the relationship betweenthe no-reflow zone and the risk zone (p=0.0085) (FIG. 8).

Regional Myocardial Blood Flow (RMBF) Data in the 4 Treatment Groups.

RMBF was measured in the ischemic region and the non-ischemic region at25 minutes of coronary artery occlusion. In the preliminary data, therewere 3 rabbits exhibiting non-ischemic values (>0.2 ml/min/g) in theischemic risk zone. This may be due to the clamp gradually looseningover the course of the occlusion. In the second part of the study, adouble-clamping method was used, and no hearts had a blood flow >0.2ml/min/g in the ischemic region. Table 16 below shows RMBF values.

TABLE 16 RMBF (ml/min/g) with 3 hearts with ischemic flow >0.20 ml/min/gexcluded Ischemic Region Non-ischemic Region Group 1 0.02 ± 0.01 2.13 ±0.12 Group 2 0.03 ± 0.01 2.63 ± 0.29 Group 3 0.05 ± 0.03 2.43 ± 0.19Control 0.02 ± 0.01 2.32 ± 0.16

D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ treatment resulted in a trend of a 21%reduction in the extent of no-reflow compared with control hearts. Therewas a significant effect of the variable “group” on the relationshipbetween the extent of the no-reflow zone and the extent of the riskregion. This signal was even stronger when treated groups were combinedand compared with control (p=0.0085).

In summary, for any given risk zone size, no-reflow was significantlyreduced by D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, a finding that has importantclinical implications. Recent studies show that worse no-reflow predictsworse LV dilation and remodeling in the chronic phase of infarction andis associated with worse prognosis, independent of changes in infarctsize. See Reffelmann T et al. Circulation 2003; 108:2911-17; Wu K et al.Circulation 1998; 97:765-72; Ndrepepa et al. J Am Coll Cardiol 2010;55:2383-89; and Bolognese et al. Circulation 2004; 109:1121-26. BecauseD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ reduces no-reflow for an average given riskzone size, we would predict that it will hasten healing (better removalof necrotic debris and better access to blood elements needed forhealing) and thus reduce LV remodeling (including reducing LV dilationand LV eccentric hypertrophy). In addition, it will likely long-termsurvival.

Other aromatic-cationic peptides as disclosed herein, for example,Phe-D-Arg-Phe-Lys-NH₂ or pharmaceutically acceptable salts thereof, suchas acetate salt or trifluoroacetate salt, could be tested and used in asimilar manner, and it is predicted that similar results would beachieved.

Example 4. Effects of D-Ara-2′,6′-Dmt-Lys-Phe-NH₂ on the Anatomic Zoneof No-Reflow Following Ischemia/Reperfusion Insult in the Brain

The effects of the aromatic-cationic peptides of the invention inprotecting a subject from an anatomic zone of no-reflow caused byischemia-reperfusion are investigated in an animal model of cerebralischemia-reperfusion injury.

Cerebral ischemia initiates a cascade of cellular and molecular eventsthat lead to brain damage. One such event is an anatomic zone ofno-reflow. Cerebral ischemia is induced by occlusion of the right middlecerebral artery for 30 min. Wild-type (WT) mice are given either salinevehicle (Veh) (ip) or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) (2-5mg/kg) at 0, 6, 24 and 48 h after ischemia. Mice are killed 3 days afterischemia. The brains are sliced transversely into 6-8 sections. Theslices are photographed under ultraviolet light to identify the regionof no-reflow. The areas of no-reflow in each slice are digitized usingImage J (supplier Rasband W S, Image J, National Institutes of Health,http://rsb.info.nih.gov/ij/). The areas in each slice are multiplied bythe weight of the slice and the results will summed in order to obtainthe mass of the no-reflow areas.

Using a mouse model of cerebral ischemia-reperfusion (20 min. occlusionof the middle cerebral artery), it is predicted that treatment of wildtype mice with D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ will result in a significantreduction in infarct volume and prevent or reduce the anatomic zone ofno-reflow. As such, the peptide D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ is effectivein reducing the incidence of no-reflow caused by ischemia-reperfusion inthe brain.

Other aromatic-cationic peptides as disclosed herein, for example,Phe-D-Arg-Phe-Lys-NH₂ or pharmaceutically acceptable salts thereof, suchas acetate salt or trifluoroacetate salt, could be tested and used in asimilar manner. It is predicted that similar results would be achievedwith the alternative aromatic-cationic peptides.

Example 5. Effects of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on the Anatomic Zoneof No-Reflow Following Ischemia/Reperfusion Insult in the Kidney

The effects of the aromatic-cationic peptides of the invention inprotecting a subject from an anatomic zone of no-reflow caused byischemia-reperfusion are investigated in an animal model of renalinjury.

Sprague Dawley rats (250-300 g) are assigned to three groups: (1) shamsurgery group without I/R; (2) I/R+ saline vehicle treatment; (3)I/R+D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) treatment.D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt) (3 mg/kg, dissolved insaline) is administered to rats 30 min before ischemia and immediatelybefore onset of reperfusion. The control rats are given saline alone onthe same schedule. Rats are anesthetized with a mixture of ketamine (90mg/kg, i.p.) and xylazine (4 mg/kg, i.p.). The left renal vascularpedicle is occluded temporarily using a micro-clamp for 30 or 45 min. Atthe end of the ischemic period, reperfusion is established by removingof the clamp. At that time, the contralateral right kidney is removed.After 24 h reperfusion, animals are sacrificed and blood samples wereobtained by cardiac puncture. Renal function is determined by blood ureanitrogen (BUN) and scrum creatinine (BioAssay Systems DIUR-500 andDICT-500).

Analysis of No-Reflow Zones, and Necrosis.

The kidneys are sliced transversely into 6-8 sections. The slices arephotographed under ultraviolet light to identify the region ofno-reflow. The areas of no-reflow in each slice are digitized usingImage J (supplier Rasband WS, Image J, National Institutes of Health,http://rsb.info.nih.gov/ij/). The areas in each slice are multiplied bythe weight of the slice and the results will summed in order to obtainthe mass of the no-reflow areas.

It is predicted that treatment with D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ preventsor reduces the anatomic zone of no-reflow after 45 min ischemia and 24 hreperfusion. For example, it is predicted that one or more of BUN, serumcreatinine and glomerular filtration rate will be improved in subjectstreated with the aromatic-cationic peptide as compared to subjects thatdid not receive the peptide treatment. As such, the peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ is effective in reducing the incidence ofno-reflow caused by ischemia-reperfusion in the kidney.

Other aromatic-cationic peptides, for example, Phe-D-Arg-Phe-Lys-NH₂ orpharmaceutically acceptable salts thereof, such as acetate salt ortrifluoroacetate salt, could be tested and used in a similar manner. Itis predicted that similar results would be achieved with the alternativearomatic-cationic peptides.

Example 6—Effects of Aromatic Cationic Peptides in Protecting Againstthe No-Reflow Phenomenon in Humans

These studies will determine whether the administration ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ at the time of revascularization would limitthe size of an anatomic zone of no-reflow that occurs upon reperfusion.For treatment of AMI, the use of mechanical recanalization of theculprit artery restores epicardial coronary blood flow to ischemicmyocardium (TIMI Flow Grade 3) in more than 90% of patients. However,these reperfusion efforts do not address the important ancillary problemof restoration of blood flow downstream at the level of the capillarybed. During or following primary PCI, microcirculatory dysfunction isobserved in 20-40% of patients. The lack of ST-segment elevationresolution after angioplasty with stenting is a marker of microvascularproblems and is associated with a poor clinical prognosis. In STEMI,failure to achieve myocardial reperfusion despite the presence of apatent coronary artery has been called the “no-reflow” phenomenon.

Study Group.

Men and women, 18 years of age or older, who present within 6 hoursafter the onset of chest pain, who have ST-segment elevation of morethan 0.1 mV in two contiguous leads, and for whom the clinical decisionis made to treat with percutaneous coronary intervention (PCI) areeligible for enrollment. Patients are eligible for the study whetherthey are undergoing primary PCI or rescue PCI. Occlusion of the culpritcoronary artery (Thrombolysis in Myocardial Infarction [TIMI] flow grade0) at the time of admission is also a criterion for inclusion.

Angiography and Revascularization.

Left ventricular and coronary angiography is performed with the use ofstandard techniques, just before revascularization. Revascularization isperformed by PCI with the use of direct stenting. Alternativerevascularization procedures include, but are not limited to, balloonangioplasty; insertion of a bypass graft; percutaneous transluminalcoronary angioplasty, and directional coronary atherectomy

Experimental Protocol.

After coronary angiography is performed but before the stent isimplanted, patients who meet the enrollment criteria are randomlyassigned to either the control group or the peptide group. Randomizationis performed with the use of a computer-generated randomizationsequence. Less than 10 min before direct stenting, the patients in thepeptide group receive an intravenous bolus injection ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (acetate salt). The peptic is dissolved innormal saline (final concentration, 25 mg per milliliter) and isinjected through a catheter that is positioned within an antecubitalvein. The patients in the control group receive an equivalent volume ofnormal saline.

No-Reflow Zone.

The primary end point is the size of the anatomic zone of no-reflow asassessed by one or more imaging techniques. Re-flow phenomenon isassessed using myocardial contrast echocardiography, coronaryangiography, myocardial blush, coronary doppler imaging,electrocardiograpy, nuclear imaging single-photon emission CT, usingthallium or technetium-99m, or PET. A 1.5-T body MRI scanner is used toperform cardiac MRI in order to assess ventricular function, myocardialedema (area at risk), microvascular obstruction and infarct size.Post-contrast delayed enhancement is used on day 4±1, day 30±3 and 6+1.5months after successful PCI and stenting to quantify infractedmyocardium. This is defined quantitatively by an intensity of themyocardial post-contrast signal that is more than 2 SD above that in areference region of remote, non-infarcted myocardium within the sameslice. Standard extracellular gadolinium-based contrast agents is usedat a dose of 0.2 mmol/kg. The 2D inversion recovery prepared fastgradient echo sequences is used at the following time points: (1) Early(approximately 2 minutes after contrast injection) for evaluation ofmicrovascular obstruction. Single shot techniques may be considered ifavailable and (2) Late (approximately 10 minutes after contrastinjection) for evaluation of infarct size.

It is predicted that administration of the D-Arg-2′,6′-Dmt-Lys-Phe-NH₂(acetate salt) at the time of reperfusion will be associated with asmaller anatomic zone of no-reflow than that seen with placebo. As such,the peptide D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ is effective in reducing theincidence of no-reflow caused by ischemia-reperfusion in the heart.

Other aromatic-cationic peptides, for example, Phe-D-Arg-Phe-Lys-NH₂, orpharmaceutically acceptable salts thereof, such as acetate salt ortrifluoroacetate salt, could be tested and used in a similar manner; itis predicted that similar results would be achieved.

REFERENCES

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EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as were apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, were apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As were understood by one skilled in the art, for any and all purposes,particularly in terms of providing a written description, all rangesdisclosed herein also encompass any and all possible subranges andcombinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as wereunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

1.-14. (canceled)
 15. A method for treating a zone of hemorrhage in amammalian subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of a peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, thereby treating the zone of hemorrhage in the subject.
 16. Themethod of claim 15, further comprising the step of performing arevascularization.
 17. The method of claim 15, wherein the subject issuffering from a zone of hemorrhage associated with a tissue selectedfrom the group consisting of cardiovascular tissue, skeletal muscletissue, cerebral tissue, and renal tissue.
 18. The method of claim 16,wherein the subject is administered the peptide prior to therevascularization procedure, after the revascularization procedure,during and after the revascularization procedure, or continuouslybefore, during, and after the revascularization procedure.
 19. Themethod of claim 18, wherein the subject is administered the peptide forat least 3 hours after the revascularization procedure, for at least 5hours after the revascularization procedure, for at least 8 hours afterthe revascularization procedure, for at least 12 hours after therevascularization procedure, or for at least 24 hours after therevascularization procedure.
 20. The method of claim 18, wherein thesubject is administered the peptide starting at least 8 hours before therevascularization procedure, starting at least 4 hours before therevascularization procedure, starting at least 2 hours before therevascularization procedure, starting at least 1 hour before therevascularization procedure, or starting at least 10 minutes before therevascularization procedure.
 21. The method of claim 15, wherein thesubject is suffering from a myocardial infarction or a stroke, or is inneed of angioplasty.
 22. The method of claim 16, wherein therevascularization procedure is selected from the group consisting ofballoon angioplasty, insertion of a bypass graft, insertion of a stent,percutaneous transluminal coronary angioplasty, and directional coronaryatherectomy.
 23. The method of claim 16, wherein the revascularizationprocedure is removal of an occlusion.
 24. The method of claim 16,wherein the revascularization procedure is administration of one or morethrombolytic agents.
 25. The method of claim 24, wherein the one or morethrombolytic agents are selected from the group consisting of tissueplasminogen activator, urokinase, prourokinase, streptokinase, acylatedform of plasminogen, acylated form of plasmin, and acylatedstreptokinase-plasminogen complex.